Bacteriophage and composition comprising same

ABSTRACT

The present invention relates to a novel bacteriophage ΦCJ24 (KCCM11462P) and a composition comprising the same as an active component. In addition, the present invention relates to a method for preventing and/or treating infectious diseases caused by avian pathogenic  E. coli  of birds by using the bacteriophage ΦCJ24 (KCCM11462P) or the composition.

INCORPORATION BY REFERENCE TO SEQUENCE LISTING

This application incorporates by reference the sequence listingsubmitted herewith. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

TECHNICAL FIELD

The present invention relates to a novel bacteriophage having a specificability to kill avian pathogenic Escherichia coli (APEC), a compositionincluding the same, and a method for preventing or treating infectiousdiseases of birds using the novel bacteriophage or the composition.

BACKGROUND ART

Escherichia coli (hereinafter also referred to as E. coli) is aGram-negative, short rod bacterium of genus Escherichia, familyEnterobacteriaceae, and one of normal flora found in intestines ofvarious animals including mammals. Most strains of Escherichia coli arenon-pathogenic and can cause opportunistic infection, but some highlypathogenic strains cause various intestinal diseases and sepsis inanimals including humans.

Among these strains of Escherichia coli, avian pathogenic E. coli (APEC)causes infection through the respiratory tract of birds such aschickens, geese, turkeys, and the like, and is known to pass into theavian body through the respiratory mucous membrane. Avian pathogenic E.coli causes diseases mostly in poultry with respect to respiratorydiseases in birds, which leads to enormous economic damage in thepoultry industry.

A bacteriophage refers to a bacterium specific virus that prevents andinhibits growth of a bacterium infected with a specific bacteriophage.As bacteriophages have stronger host specificity than antibiotics, andrecent emergence of bacteria resistant to antibiotics and residualantibiotics in animals are growing problems, application ofbacteriophages has drawn great interest.

Studies on bacteriophages have been actively performed in manycountries, and there has been an increasing tendency to obtain approvalfrom the Food and Drug Administration (FDA) for compositions usingbacteriophages in addition to patent applications for bacteriophages.

However, bacteriophage related technologies for prevention and/ortreatment of infectious diseases, which are important issues in theaviculture industry including poultry farming, due to avian pathogenicEscherichia coli are still insufficient, and therefore, there is a needfor such bacteriophages and development of relevant technologies.

DISCLOSURE Technical Problem

As a result of earnest investigation aimed at overcoming the emergenceof bacteria resistant to antibiotics and residual antibiotics in animalsand at effectively preventing and treating infectious diseases of birds,the present inventors isolated a novel bacteriophage ΦCJ24 (KCCM11462P)having a specific ability to kill avian pathogenic Escherichia colicausing respiratory diseases of poultry from natural sources.

In addition, the present inventors identified morphological,biochemical, and genetic properties of the novel bacteriophage,confirmed that the bacteriophage has excellent acid resistance, heatresistance, and drying resistance, and developed antibiotics,disinfectants, additives for feeds, and other compositions using thebacteriophage, a composition for preventing or treating infectiousdiseases in birds, and a method for preventing or treating diseasesusing the same.

It is an object of the present invention to provide a novelbacteriophage ΦCJ24 (KCCM11462P) having a specific ability to kill avianpathogenic Escherichia coli.

It is another object of the present invention to provide a compositionfor preventing and/or treating infectious diseases caused by avianpathogenic Escherichia coli, including the bacteriophage ΦCJ24(KCCM11462P) as an active ingredient.

It is a further object of the present invention to provide antibiotics,additives for feeds, additives for drinking water, feeds, drinkingwater, disinfectants or detergents, including the bacteriophage ΦCJ24(KCCM11462P) as an active ingredient.

It is yet another object of the present invention to provide a methodfor preventing and/or treating infectious diseases caused by avianpathogenic Escherichia coli using the bacteriophage ΦCJ24 (KCCM11462P)or the composition including the bacteriophage ΦCJ24 (KCCM11462P) as anactive ingredient.

Technical Solution

One aspect of the present invention provides a novel bacteriophage ΦCJ24(KCCM11462P) having a specific ability to kill avian pathogenicEscherichia coli.

Another aspect of the present invention provides a composition forpreventing or treating infectious diseases caused by avian pathogenicEscherichia coli, including the bacteriophage ΦCJ24 (KCCM11462P) as anactive ingredient.

A further aspect of the present invention provides antibiotics,additives for feeds, additives for drinking water, feeds, drinkingwater, disinfectants or detergents, including the bacteriophage ΦCJ24(KCCM11462P) as an active ingredient.

Yet another aspect of the present invention provides a method forpreventing or treating infectious diseases caused by avian pathogenicEscherichia coli, including: administering the bacteriophage ΦCJ24(KCCM11462P) or the composition including the bacteriophage ΦCJ24(KCCM11462P) as an active ingredient to birds.

Advantageous Effects

The bacteriophage ΦCJ24 (KCCM11462P) according to the present inventionhas an effect of having a specific ability to kill avian pathogenicEscherichia coli.

Further, the bacteriophage ΦCJ24 (KCCM11462P) according to the presentinvention has excellent acid resistance, heat resistance, and dryingresistance, and thus can be employed not only as an agent for preventingor treating infectious diseases caused by avian pathogenic Escherichiacoli at various ranges of temperature, pH, and dry conditions but alsoas antibiotics, additives for feeds, additives for drinking water,feeds, drinking water, disinfectants, detergents, and the like,including the bacteriophage ΦCJ24 (KCCM11462P) as an active ingredient.

Further, the present invention provides the bacteriophage ΦCJ24(KCCM11462P) or antibiotics including the same as an active ingredient,and the antibiotics have effects in that the antibiotics havespecificity for avian pathogenic Escherichia coli as compared to priorantibiotics and thus selectively kill specific pathogenic bacteriawithout killing beneficial bacteria; and that the antibiotics do notinduce antibiotic resistance, resulting in extension of lifetime ofproducts as compared to prior antibiotics.

Further, the present invention has effects of preventing or treatinginfectious diseases caused by avian pathogenic Escherichia coli byadministering the bacteriophage ΦCJ24 (KCCM11462P) or the compositionincluding the bacteriophage ΦCJ24 (KCCM11462P) as an active ingredientto birds.

DESCRIPTION OF DRAWINGS

FIG. 1 is an electron microscope image of a novel bacteriophage ΦCJ24(KCCM11462P) (hereinafter referred to as ‘ΦCJ24’).

FIG. 2 shows results of pulsed field gel electrophoresis (PFGE) of anovel bacteriophage ΦCJ24.

FIG. 3 shows results of sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) of a novel bacteriophage ΦCJ24.

FIG. 4 is a graph depicting results of acid resistance experiment of anovel bacteriophage ΦCJ24.

FIG. 5 is a graph depicting results of heat resistance experiment of anovel bacteriophage ΦCJ24.

FIG. 6 is a graph depicting results of drying resistance experiment of anovel bacteriophage ΦCJ24.

EMBODIMENTS

Hereinafter, embodiments of the present invention will be described inmore detail. Description of details apparent to a person having ordinaryknowledge in the art will be omitted herein.

One embodiment of the present invention provides a novel bacteriophageΦCJ24 (KCCM11462P) (hereinafter referred to as ‘ΦCJ24’) having aspecific ability to kill avian pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli refers to Escherichia coli that istransmitted through the respiratory tract of birds such as chickens,geese, turkeys, and the like, and that can cause infectious diseases ofbirds, specifically avian colibacillosis. Specifically, avian pathogenicEscherichia coli penetrates into the body of birds through the mucousmembrane of the respiratory tract, and causes various diseases such assepsis, granuloma, air sacculitis, salpingitis, arthritis, and the like.Avian pathogenic Escherichia coli is a Gram-negative bacillus just likegeneral Escherichia coli, has peritrichous flagella for motility, and isan aerobic or facultative anaerobic bacterium which decomposes lactoseand fructose to generate acids and gases.

Avian pathogenic Escherichia coli grows well on common media and iscapable of growing at a temperature of about 7° C. to about 48° C. withideal growth temperature ranging from about 35° C. to about 37° C.Specifically, at around 42° C. which is close to body temperature ofbirds, expression of pathogenic factors is effectively performed.Further, avian pathogenic Escherichia coli can grow at pH ranging frompH 4.5 to pH 9.0.

A bacteriophage is a bacteria-specific virus capable of infecting aspecific bacterium and inhibiting growth of the bacterium, and is avirus including single or double-stranded deoxyribonucleic acid (DNA) orribonucleic acid (RNA) as a genetic material.

Specifically, the bacteriophage ΦCJ24 according to the embodiment of thepresent invention is a bacteriophage that has species specificity ofselectively infecting avian pathogenic Escherichia coli andmorphologically belongs to morphotype B1 Siphoviridae having anicosahedral capsid and a long non-contractile tail (see FIG. 1).Homology between a nucleotide sequence of the bacteriophage ΦCJ24 anddecoded nucleotide sequences of other bacteriophages is compared andresults are shown in Table 1. The bacteriophage ΦCJ24 shows stable acidresistance at pH 3.5 to pH 11.0 without losing activity (FIG. 4), and interms of heat resistance, the bacteriophage ΦCJ24 shows activity declineof about 1 log or less when exposed to 50° C. or more for two hours(FIG. 5). In terms of drying resistance, the bacteriophage ΦCJ24 showsactivity decline of about 1 log when dried at 60° C. for two hours (FIG.6). Partial DNA nucleotide sequences of the bacteriophage ΦCJ24 are setforth in SEQ ID NOs: 1 and 2 of Sequence List.

The bacteriophage ΦCJ24 is a novel bacteriophage isolated by the presentinventor, and was deposited at the Korean Culture Center ofMicroorganisms (KCCM) (361-221, Hongje l-dong, Seodaemun-gu, Seoul,Korea) on Oct. 25, 2013 under accession number KCCM 11462P.

Another embodiment of the present invention provides a composition forpreventing or treating infectious diseases caused by avian pathogenicEscherichia coli, including the bacteriophage ΦCJ24 as an activeingredient.

Since the bacteriophage ΦCJ24 exhibits antibacterial activity capable ofspecifically killing avian pathogenic Escherichia coli, thebacteriophage ΦCJ24 can be utilized in prevention or treatment ofdiseases caused by infection with avian pathogenic Escherichia coli.Examples of infectious diseases caused by avian pathogenic Escherichiacoli include avian colibacillosis, without being limited thereto.

Herein, the term “avian colibacillosis” refers to a disease occurring inthe respiratory tract of birds due to infection with pathogenicEscherichia coli, and symptoms thereof include air sacculitis,perihepatitis, peritonitis, pericarditis, salpingitis, omphalitis,osteomyelitis or septicemia, thereby causing growth delay and mortalityof infected birds.

Herein, the term “preventing” or “prevention” refers to all actions toinhibit the diseases or delay occurrence of the diseases byadministering the bacteriophage ΦCJ24 and/or the composition includingthe bacteriophage ΦCJ24 as an active ingredient to an animal.

Herein, the term “treating” or “treatment” refers to all actions toimprove or ameliorate symptoms of infectious diseases by administeringthe bacteriophage ΦCJ24 and/or the composition including thebacteriophage ΦCJ24 as an active ingredient to an animal.

The composition for preventing or treating infectious diseases caused byavian pathogenic Escherichia coli according to this embodiment mayinclude the bacteriophage ΦCJ24 in amounts of 5×10² pfu/ml to 5×10¹²pfu/ml, specifically, 1×10⁶ pfu/ml to 1×10¹⁰ pfu/ml.

The composition for preventing or treating infectious diseases caused byavian pathogenic Escherichia coli according to this embodiment mayfurther include pharmaceutically acceptable carriers, and may beformulated with the carriers to provide foods, medicines, additives forfeeds or additives for drinking water, and the like. Herein, the term“pharmaceutically acceptable carriers” refers to carriers or diluentsthat do not stimulate an organism and do not inhibit biological activityand properties of administered compounds.

Types of carriers applicable to this embodiment are not particularlylimited and any pharmaceutically acceptable carriers commonly used inthe art may be utilized. Examples of the carriers may include saline,distilled water, Ringer's solution, buffered saline, an albumininjection solution, a dextrose solution, a maltodextrin solution,glycerol, and ethanol, without being limited thereto. These may be usedalone or in combination thereof.

Furthermore, as needed, other common additives such as antioxidants,buffered solutions and/or cytostatics may be added to the compositionaccording to the present invention, and diluents, dispersants,surfactants, binders and/or lubricants may be further added to thecomposition according to the present invention to formulate injectableformulations such as aqueous solutions, suspensions and emulsions,pills, capsules, granules, and tablets.

Methods for administering the composition for preventing or treatinginfectious diseases caused by avian pathogenic Escherichia coliaccording to this embodiment are not particularly limited, and anymethods commonly used in the related art may be used. One example of theadministration method may include oral administration or parenteraladministration.

Examples of dosage forms for oral administration may include troches,lozenges, tablets, water soluble suspensions, oil-based suspensions,formulated powder, granules, emulsions, hard capsules, soft capsules,syrups, or elixirs, and the like.

In order to formulate the composition according to this embodiment intodosage forms such as tablets or capsules, binders such as lactose,saccharose, sorbitol, mannitol, starches, amylopectin, cellulose andgelatin; excipients such as dicalcium phosphate; disintegrators such ascorn starch and sweet potato starch; lubricants such as magnesiumstearate, calcium stearate, sodium stearyl fumarate and polyethyleneglycol wax may be further included, and for capsule formulation, liquidcarriers such as fatty oils may be further included in addition to theaforementioned substances.

Methods for parenterally administering the composition of thisembodiment may include, for example, intravenous injection,intraperitoneal administration, intramuscular administration,subcutaneous administration, and topical administration, and a method ofapplying or spraying the composition according to the present inventionto an affected region, without being limited thereto.

In order to formulate parenteral dosage forms, for example, thecomposition of this embodiment may be formulated into dosage forms forinjection such as subcutaneous injection, intravenous injection andintramuscular injection; suppositories; or dosage forms for sprayingsuch as aerosols so as to permit inhalation through inhalers, withoutbeing limited thereto. In order to formulate dosage forms for injection,the composition of this embodiment may be mixed with stabilizers orbuffering agents in water to prepare solutions or suspensions, which areformulated into dosage forms for unit administration such as ampoules orvials. When the composition is formulated into dosage forms for sprayingsuch as aerosols, the composition may be formulated with propellants andthe like together with additives such that a concentrate dispersed inwater or wetted powder is dispersed therein.

Suitable amounts of applying, spraying or administering the compositionfor preventing or treating infectious diseases caused by avianpathogenic Escherichia coli according to this embodiment may differaccording to factors such as age, body weight and sex of animals, degreeof disease symptoms, ingested foods, rate of excretion, and the like inaddition to a method for formulating the composition, an administrationmethod, administration time and/or routes for administration, and agenerally skilled veterinarian can easily determine and prescribe doseamounts effective for intended treatment.

A further embodiment of the present invention provides antibioticsincluding the bacteriophage ΦCJ24 as an active ingredient.

Herein, the term “antibiotics” refers to a preparation that isadministered to animals including humans in medicine form and exhibitsefficacy of sterilizing bacteria, and is used as a general term forantiseptics, germicides and antibacterial agents.

Antibiotics of this embodiment including the bacteriophage ΦCJ24 as anactive ingredient have effects in that the antibiotics have specificityfor avian pathogenic Escherichia coli as compared to typical antibioticsand thus kill specific pathogenic bacteria, but not beneficial bacteria;and in that the antibiotics do not induce drug resistance, causingextension of lifetime of products as compared to typical antibiotics.

Yet another embodiment of the present invention provides an additive foravian feeds or avian drinking water, which includes the bacteriophageΦCJ24 as an active ingredient.

Birds as a subject to which the additives for avian feeds or theadditives for avian drinking water are applied are not particularlylimited, but birds in this embodiment are particularly poultry.

Herein, poultry is a generic name for animals belonging to birds amonglivestock. Poultry is not particularly limited, and may comprise atleast one selected from the group consisting of chickens, geese,turkeys, and the like.

The additives for avian feeds or the additives for avian drinking watermay be used by separately preparing additives for feeds or additives foravian drinking water using the bacteriophage ΦCJ24 or the compositionincluding the same and mixing feeds or drinking water with theadditives, or directly adding the bacteriophage ΦCJ24 or the compositionincluding the same in a process of preparing feeds or drinking water.

The bacteriophage ΦCJ24 or the composition including the bacteriophageΦCJ24 as an active ingredient used in the form of additives for feeds oradditives for drinking water according to this embodiment may be aliquid form or a dried form, for example, a dried powder form.

For example, the bacteriophage ΦCJ24 according to the present inventionis mixed in powder form in amounts of 0.05% by weight (wt %) to 10 wt %,specifically 0.1 wt % to 2 wt %, based on the weight of additives forfeeds.

Methods for drying the additives for feeds or additives for drinkingwater according to this embodiment to yield dried powder are notparticularly limited, and any methods commonly used in the related artmay be utilized. Examples of the drying method may include air drying,natural drying, spray drying, and lyophilization, without being limitedthereto. These methods may be used alone or in combination thereof.

The additives for feeds or additives for drinking water according tothis embodiment may further include other non-pathogenic microorganisms.The microorganisms may be selected from the group consisting of Bacillussp. such as Bacillus subtilis capable of producing proteases, lipasesand/or glycosyltransferase s; lactic acid bacteria such as Lactobacillussp. having physiological activity and organic material decomposingcapability under anaerobic conditions like the stomach of cattle;filamentous bacteria such as Aspergillus oryzae having effects of weightgain in animals, increase in milk production, and increase ofdigestion-absorption rate of feeds; and yeasts such as Saccharomycescerevisiae and the like. These microorganisms may be used alone or incombination thereof.

The additives for feeds or additives for drinking water according tothis embodiment including the bacteriophage ΦCJ24 as an activeingredient may further include other additives as needed. Examples ofusable additives may include binders, emulsifiers, and preservativesadded for prevention of quality deterioration of feeds or drinkingwater; amino acid, vitamin, enzyme, probiotics, flavoring agents,non-protein nitrogen compounds, silicate, buffering agents, coloringagents, extracting agents or oligosaccharides that are added in order toincrease utility of feeds or drinking water; and other supplements tofeeds, and the like. These additives may be used alone or in combinationthereof.

The additives for feeds according to the present invention may bepresent in amounts of 0.05 parts by weight to 10 parts by weigh,specifically 0.1 parts by weight to 2 parts by weight, based on 100parts by weight of feeds. The additives for drinking water according tothe present invention may be present in amounts of 0.0001 parts byweight to 0.01 parts by weight, specifically 0.001 parts by weight to0.005 parts by weight, based on 100 parts by weight of drinking water.Within these ranges, the additives allow activity of the bacteriophageΦCJ24 against avian pathogenic Escherichia coli to be sufficientlydisplayed.

Yet another embodiment of the present invention provides feeds ordrinking water prepared by adding the additives for feeds or theadditives for drinking water including the bacteriophage ΦCJ24 as anactive ingredient to feeds or drinking water, or directly adding thebacteriophage ΦCJ24 thereto.

Feeds used in this embodiment are not particularly limited, and anyfeeds commonly used in the related art may be used. Examples of thefeeds may include vegetable feeds such as grains, root vegetables, foodprocessing byproducts, algae, fibers, pharmaceutical byproducts, oilsand fats, starches, residues or byproducts of grain, and the like; andanimal feeds such as proteins, inorganic substances, oils and fats,minerals, single cell proteins, and animal planktons or foods. Thesefeeds are used alone or in combination thereof.

Drinking water used in this embodiment is not particularly limited, andany drinking water commonly used in the related art may be used.

Yet another embodiment of the present invention provides disinfectantsor detergents including the bacteriophage ΦCJ24 as an active ingredient.Dosage forms of the disinfectants or detergents are not particularlylimited, and any dosage forms commonly used in the related art may beused.

In order to remove avian pathogenic Escherichia coli, the disinfectantsmay be sprayed to habitats of birds, slaughterhouses, dead regions,kitchens, and cooking equipment, without being limited thereto.

The detergents may be used to wash a surface of the dermis or body partsof birds that are exposed to or can be exposed to avian pathogenicEscherichia coli, without being limited thereto.

Yet another embodiment of the present invention provides a method forpreventing or treating infectious diseases caused by avian pathogenicEscherichia coli using the bacteriophage ΦCJ24 or the compositionincluding the bacteriophage ΦCJ24 as an active ingredient.

Specifically, the prevention method or treatment method of thisembodiment includes administering a pharmaceutically effective amount ofthe bacteriophage ΦCJ24 or the composition including the bacteriophageΦCJ24 as an active ingredient to birds that are exposed to or can beexposed to avian pathogenic Escherichia coli. Suitable total amounts ofthe bacteriophage ΦCJ24 or the composition including the same per daymay be determined by a physician within proper medicinal judgment, asapparent to those skilled in the art.

A concrete pharmaceutically effective amount of the bacteriophage ΦCJ24or the composition including the bacteriophage ΦCJ24 as an activeingredient to certain birds may be determined by taking into account thesorts and degree of reaction to achieve, age, body weight, generalhealth condition, sex or diet of corresponding individuals,administration time and administration routes of bacteriophage ΦCJ24 ora composition including the same, and secretion rate of the composition,treatment period, and the like, and may differ depending upon variousfactors and similar factors well known in the field of medicineincluding ingredients of medicines that are used simultaneously or atdifferent times.

The bacteriophage ΦCJ24 or the composition including the bacteriophageΦCJ24 as an active ingredient may be administered in the form of apharmaceutical preparation to birds by intranasal spraying, or directlyadded to avian feeds or drinking water so as to be digested, and may bemixed in the form of additives for feeds or additives for drinking waterwith feeds or drinking water and then administered.

Routes and methods for administration of the bacteriophage ΦCJ24 or thecomposition including the bacteriophage ΦCJ24 as an active ingredientare not particularly limited, and the administration may be realized byany routes and methods so long as the administration allows thebacteriophage ΦCJ24 or the composition including the same to reachdesired tissues. Namely, the bacteriophage ΦCJ24 or the compositionincluding the bacteriophage ΦCJ24 as an active ingredient may beadministered by various oral or parenteral routes, and examples ofadministration may include oral, rectal, topical, intravenous,intraperitoneal, intramuscular, intra-arterial, trans-dermal,intranasal, and inhalation, without being limited thereto.

Hereinafter, the present invention will be described in more detail withreference to a preferred example. It should be understood that theseexamples are not to be construed in any way as limiting the presentinvention.

Example 1 Isolation of Bacteriophage that Infects Avian PathogenicEscherichia coli Example 1-1

Bacteriophage Screening and Single Bacteriophage Isolation

50 ml of a specimen obtained from chicken feces collected around apoultry farm in Chengwon-gun, Chungcheong Province was centrifuged at4,000 rpm for 10 minutes, and the resulting supernatant was filteredthrough a 0.45 μm filter to prepare a specimen liquid, which in turn wasused to perform a soft agar overlay method. The soft agar overlay methodrefers to a method of observing bacteriophage lysis using a host cellgrowing on top-agar (attached to a solid medium using 0.7% agar).

Specifically, 150 μl of a shaking culture solution (OD₆₀₀=2) of avianpathogenic Escherichia coli (E10-4) obtained from the Department ofVeterinary Medicine of Konkuk University and 2 ml of 10×LB medium (10g/l of tryptophan; 5 g/l of yeast extract; 10 g/l of NaCl) were mixedwith 18 ml of the filtered specimen liquid, followed by culturing at 30°C. for 18 hours, and the resulting cultured solution was centrifuged at4,000 rpm for 10 minutes, and the resulting supernatant was filteredthrough a 0.45 μm filter. Subsequently, a mixed solution consisting of 3ml of 0.7% (w/v) agar and 150 μl of a shaking culture solution (OD₆₀₀=2)of avian pathogenic Escherichia coli (E10-4) was poured and solidifiedon an LB medium plate, to which 10 μl of the specimen liquid was addeddropwise, followed by culturing at 30° C. for 18 hours, therebyidentifying formation of plaques.

Since it is known that one sort of bacteriophage is present per plaque,the inventors tried to isolate single bacteriophages from the formedplaques. Specifically, 400 μl of SM solution (5.8 g/l of NaCl; 2 g/l ofMgSO₄7H₂O₂; 50 ml of 1M Tris-HCl (pH 7.5)) was added to the plaques andleft at room temperature for 4 hours, thereby obtaining a bacteriophagesolution.

Subsequently, 100 μl of the bacteriophage solution was mixed with 12 mlof 0.7% (w/v) agar and 500 μl of a shaking culture solution (OD₆₀₀=2) ofavian pathogenic Escherichia coli (E10-4), which was used to perform asoft agar overlay method using an LB medium plate having a diameter of150 mm wherein cultivation was performed until the bacteriophage wascompletely lysed. After completion of cultivation, 15 ml of SM solutionwas added to the LB medium plate and left at room temperature for 4hours, thereby obtaining a bacteriophage solution.

To the obtained solution, 1% (v/v) chloroform was added and mixed for 10minutes, followed by centrifugation at 4,000 rpm for 10 minutes, therebyobtaining a supernatant, which in turn was filtered through a 0.45 μmfilter, thereby obtaining a final specimen.

Example 1-2

Large Scale Culture and Purification of Bacteriophage

The bacteriophage obtained in Example 1-1 was cultured at large scaleusing avian pathogenic Escherichia coli (E10-4), and then thebacteriophage was purified therefrom.

Specifically, avian pathogenic Escherichia coli (E10-4) was shakingcultured, and inoculated at 1.0×10¹⁰ cfu, followed by centrifuging at4,000 rpm for 10 minutes, and re-suspending in 4 ml of SM solution. Tothis, the bacteriophage was added at 1.0×10⁶ pfu with multiplicity ofinfection (MOI) of 0.0001, and then left at room temperature for 20minutes.

Next, 150 ml of LB medium was inoculated therewith, and cultured at 30°C. for 6 hours. After completion of cultivation, chloroform was added toa volume of 1% (v/v) of the final volume, followed by stirring for 20minutes, to which DNase I and RNase A as restriction enzymes were addedin a final concentration of 1 μg/ml, respectively, and left at 30° C.for 30 minutes. Subsequently, sodium chloride and polyethylene glycolwere added to a final concentration of 1M and 10% (w/v), respectively,and left at 4° C. for 3 hours, followed by centrifuging at 4° C. and12,000 rpm for 20 minutes, thereby obtaining a precipitate.

The obtained precipitate was suspended in 5 ml of SM solution and thenleft at room temperature for 20 minutes, 4 ml of chloroform was addedthereto with stirring, followed by centrifugation at 4° C. with 4,000rpm for 20 minutes, thereby obtaining a supernatant. The supernatant wasfiltered through a 0.45 μm filter, followed by ultracentrifugation(35,000 rpm, 1 hour, 4° C.) using a glycerol density gradient method(density: 40%, 5% glycerol), thereby purifying a bacteriophage.

The present inventors isolated a bacteriophage having a specific abilityto kill avian pathogenic Escherichia coli from samples collected fromchicken feces on farms, which was designated as “Bacteriophage ΦCJ24”and deposited at the Korean Culture Center of Microorganisms (KCCM)(361-221 Hongje l-dong, Seodaemun-gu, Seoul, Korea) on Oct. 25, 2013under accession number KCCM 11462P.

Example 2

Morphology Observation of ΦCJ24

The bacteriophage ΦCJ24 purified in Example 1 was diluted in 0.01%gelatin solution, and then fixed with a 2.5% glutaraldehyde solution.The resulting bacteriophage was added dropwise to a carbon-coated micaplate (ca. 2.5 mm×2.5 mm), acclimated for 10 minutes, and then washedwith distilled water.

Subsequently, the carbon film was mounted on a copper grid, and stainedwith 4% uranyl acetate for 60 seconds, dried, and examined under atransmission electron microscope (JEM-1011, 80 kV, magnification of×200,000) (FIG. 1).

FIG. 1 is a transmission electron microscope image of bacteriophageΦCJ24, in which the bacteriophage ΦCJ24 had morphologicalcharacteristics of an icosahedral capsid with a long non-contractiletail, indicating that the bacteriophage belongs to morphotype B1Siphoviridae.

Example 3

Total Genomic DNA Size Analysis of ΦCJ24

Genomic DNA was extracted from the bacteriophage ΦCJ24 purified inExample 1.

Specifically, to a cultured solution of the purified bacteriophageΦCJ24, 20 mM ethylenediaminetetraacetic acid (EDTA), 50 μg/ml protease Kand 0.5% (w/v) sodium dodecyl sulfate (SDS) were added and left at 50°C. for one hour, to which an equal amount of phenol (pH 8.0) was addedwith stirring, followed by centrifugation at room temperature and 12,000rpm for 10 minutes, thereby obtaining a supernatant.

The supernatant was mixed with an equal amount of PC(phenol:chloroform=1:1), followed by centrifugation at room temperatureand 12,000 rpm for 10 minutes, thereby obtaining a supernatant. Thesupernatant was mixed with an equal amount of chloroform, followed bycentrifugation at room temperature and 12,000 rpm for 10 minutes,thereby obtaining a supernatant. The supernatant was mixed with 10%(v/v) of 3M sodium acetate based on the total volume, followed by theaddition of 2 volumes of 95% cold ethanol, mixing, and standing at −20°C. for 1 hour.

Subsequently, the resulting substance was centrifuged at 0° C. and12,000 rpm for 10 minutes, from which a supernatant was removed toobtain a precipitate, which was dissolved in 50 μl of TE bufferedsolution (Tris-EDTA, pH 8.0). The extracted DNA was diluted 10 fold, andthen concentration of DNA was determined by measuring absorbance atOD₂₆₀.

Next, 1 μg of DNA was loaded on a 1% PFGE (pulsed field gelelectrophoresis) agarose gel, and developed using BIORAD PFGE SYSTEM NO.7 PROGRAM (size ranging from 25 kb to 100 kb; switch time ramp 0.4seconds to 2.0 seconds, linear shape; forward voltage, 180 V; reversevoltage, 120 V) at room temperature for 20 hours (FIG. 2).

FIG. 2 is an electrophoresis gel photograph of genomic DNA of thebacteriophage ΦCJ24, and it could be seen that the genomic DNA size ofthe bacteriophage ΦCJ24 was about 53 kbp.

Example 4

Protein Pattern Analysis of ΦCJ24

15 μl of purified bacteriophage ΦCJ24 solution (10¹¹ pfu/ml titer) wasmixed with 3 μl of 5×SDS sample solution, and then boiled for 5 minutesto perform 12% SDS-PAGE (FIG. 3).

FIG. 3 is an electrophoresis photograph of SDS-PAGE results performed onthe bacteriophage ΦCJ24, and it could be seen that main proteins had asize of about 10.3 kDa, about 12.5 kDa, about 15.1 kDa, about 43 kDa,about 49.3 kDa, about 60.4 kDa and about 94.9 kDa.

Example 5

Analysis of Genetic Properties of ΦCJ24

In order to determine genetic properties of the bacteriophage ΦCJ24purified in Example 1, DNA of the bacteriophage ΦCJ24 was analyzed usingan FLX Titanium Sequencer (Roche) as a gene analyzer. Genes wererecombined using GS and de novo assembler software (Roche) by MacrogenInc. Open reading frame was identified using GeneMark.hmm, Glimmer v3.02and FGENESB software. Open reading frame was annotated using BLASTP andInterProScan program.

Nucleotide sequence of the bacteriophage ΦCJ24 showed similarity tonucleotide sequence of previously reported bacteriophages (Escherichiaphage phiEB49, Escherichia phage KBN21), but it could be seen that therewere no bacteriophages in which all fragments 100% coincide.Accordingly, it could be seen that the bacteriophage was a novelisolated bacteriophage.

The following Table 1 shows comparison results between nucleotidesequence of the bacteriophage ΦCJ24 and decoded nucleotide sequence ofthe prior reported bacteriophage in the art.

TABLE 1 Score Identities Query Subject E- Match/ Pct. Name Length StartEnd Description Bit Value Total (%) SEQ 50547 25529 27458 Escherichia1283 0 1638/ 83 ID phage 1959 NO: phiEB49, 1 complete genome SEQ 8358 48358 Escherichia 16360 0 8330/ 99 ID phage 8355 NO: KBNP21, 2 completegenome

DNA of the prepared bacteriophage ΦCJ24 was analyzed using a DNAsequencer and partial results of the analyzed nucleotide sequence areset forth in SEQ ID NOs: 1 and 2.

Example 6

pH Stability of ΦCJ24

In order to identify whether the bacteriophage ΦCJ24 can maintainstability at low pH like stomach conditions, stability of thebacteriophage ΦCJ24 was examined at various pH (pH 2.5, 3.0, 3.5, 4.0,5.5, 6.5, 7.0, 8.0, 9.0, 10.0 and 11.0).

For the experiment, various pH solutions (sodium acetate buffersolutions (pH 4.0, pH 4.5, pH 5.0, pH 5.5), sodium citrate buffersolutions (pH 2.5, pH 3.0 and pH 3.5), sodium phosphate buffer solutions(pH 6.5 and pH 7.0), and Tris-HCl solutions (pH 8.0, pH 9.0, pH 10.0 andpH 11.0) were prepared at a concentration of 0.2M.

180 μl of each pH solution was mixed with 20 μl of a bacteriophagesolution with 2.0×10¹¹ PFU/ml titer to allow each pH solution to have aconcentration of 1M, and then the resulting solution was left at roomtemperature for 2 hours. For a control group, 20 μl of a bacteriophagesolution with 2.0×10¹¹ PFU/ml titer was mixed with 180 μl of SM solutionby the same method, and the resulting solution was left at roomtemperature for 2 hours. Thereafter, the solutions were seriallydiluted, and 10 μl of each of solutions in each dilution step wascultured by the soft agar overlay method at 30° C. for 18 hours todetermine bacteriophage titer based on whether the bacteriophage waslysed (FIG. 4).

FIG. 4 shows experimental results of acid resistance of thebacteriophage ΦCJ24. In FIG. 4, it could be seen that the bacteriophageΦCJ24 did not lose its activity and maintained stability from pH 3.5 topH 11.0, as compared with the control group.

Example 7

Heat Stability of Bacteriophage ΦCJ24

If bacteriophages are formulated into additives for feeds among dosageforms of bacteriophages, heat can be generated during formulationprocedures, and thus, the following experiment was performed in order todetermine heat stability of bacteriophages.

Specifically, 100 μl of bacteriophage ΦCJ24 solution with 1.65×10¹¹PFU/ml was left at 37° C., 45° C., 53° C. and 60° C. for 10 minutes, 30minutes, 60 minutes and 120 minutes, respectively. Thereafter, theresulting experimental culture solution was serially diluted, 10 μl ofeach of solutions in each dilution step was cultured by the soft agaroverlay method at 30° C. for 18 hours to determine bacteriophage titerbased on whether the bacteriophage was lysed (FIG. 5).

FIG. 5 shows experimental results of heat resistance of bacteriophageΦCJ24. As shown in FIG. 5, it could be seen that bacteriophage ΦCJ24showed activity loss of about 1 log or less until bacteriophage ΦCJ24was exposed to 53° C. for 120 minutes, and activity decline was observedas time went by when bacteriophage ΦCJ24 was exposed to 60° C.

Example 8

Drying Stability of Bacteriophage ΦCJ24

If bacteriophages are formulated into additives for feeds among dosageforms of bacteriophages, bacteriophages can be dried during formulationprocedures, and thus, the following experiment was performed in order todetermine stability of bacteriophages against drying conditions.

Based on the results from heat resistance experiment, drying experimentwas performed using a SpeedVac concentrator. 200 μl of bacteriophageΦCJ24 solution with 2.5×10¹⁰ PFU/ml was dried at 60° C. under vacuum for2 hours, and the resulting pellets were introduced to 200 μl of SMsolution, followed by completely re-suspending at 4° C. for one day,thereby measuring titers (FIG. 6).

As shown in FIG. 6, it could be seen that after drying, as compared withinitial titers and relative stability, bacteriophage ΦCJ24 showedactivity loss of about 1 log when bacteriophage ΦCJ24 was dried at 60°C. for 2 hours.

Example 9

Examination of Infection Range of Bacteriophage ΦCJ24 on a Wild-TypeIsolated Strain, Avian Pathogenic Escherichia coli

Lytic activity of bacteriophage ΦCJ24 was tested for 46 strains of thewild-type avian pathogenic Escherichia coli isolated by College ofVeterinary Medicine, Konkuk University (KU), 10 strains of avianpathogenic Escherichia coli isolated by Korea Animal and PlantQuarantine Agency (KAPQA), 7 strains of avian pathogenic Escherichiacoli isolated by College of Veterinary Medicine, Chonbuk NationalUniversity (CNU), and 26 strains of disease-diagnosed avian pathogenicEscherichia coli isolated by Komipharm farm (KF) in addition to avianpathogenic Escherichia coli (E10-4) used in the present experiment.

Specifically, 150 μl of a shaking culture solution of each strain(OD₆₀₀=2) was mixed, and 10 μl of bacteriophage ΦCJ24 solution with 10⁹pfu/ml titer was dropped thereto and cultured by the soft agar overlaymethod at 30° C. for 18 hours, and then plaque formation was examined(Table 2).

The results are shown in Table 2.

TABLE 2 KU Sero- No. strains typing ΦCJ24 1 E09-1 2 E09-2 3 E09-3 0 4E09-4 5 E09-5 6 E09-6 O-78 0 7 E09-7 8 E09-8 O-78 0 9 E09-9 O-78 0 10E09-10 11 E09-11 O-78 0 12 E09-12 O-125 13 E09-13 14 E09-14 15 E09-15 016 E09-16 17 E09-17 18 E09-18 19 E09-19 20 E09-20 21 E09-21 22 E09-22 23E09-23 0 24 E09-24 25 E09-25 26 E09-26 27 E09-27 0 28 E09-28 29 E09-2930 E09-30 0 31 E09-31 32 E09-32 0 33 E09-33 34 E09-34 0 35 E09-35(297)O-78 0 36 E09-36(343) 37 E09-37(343) 38 E09-38(343) 39 E09-39(353) 40E09-40(353) 41 E09-41(376) 42 E09-42(376) 43 E10-2 O-1 0 44 E10-3 O-78 045 E10-4 O-78 0 46 E10-5 O-78 0 47 E10-6 KAPQA Sero- No. strains TypingΦCJ24 48 O6Q-035 O-78 0 49 O6D-044 O-78 0 50 O6Q-140 O-78 0 51 O7D-001O-78 0 52 O7D-022 O-78 0 53 07Q-039 O-78 0 54 KWU-02 O-78 0 55 KWU-32O-78 0 56 KWU-33 O-78 0 57 KWU-43 O-78 0 Sero- No. CNU Strains TypingΦCJ24 58 A12-MRA-076-{circle around (1)} 59 A10-LSf-005 60 A11-LSF-04361 A12-MRA-076-{circle around (2)} 62 D12-JW-058 63 A12-LSF-083 O-78 064 A12-MRA-076-{circle around (3)} Sero- No. KF Strains typing ΦCJ24 6512-001-3 66 12-053 67 12-055 68 12-086-1 O-78 0 69 12-086-2 O-78 0 7012-096-3 71 12-175 72 12-187 O-78 0 73 12-211-5 74 12-220-4 75 12-220-676 12-248 77 12-261-1 78 12-266 79 12-274-1 80 12-275-2 O-78 0 8112-286-2 82 12-300 O-78 0 83 12-303-2 O-78 0 84 12-304-3 85 12-299-1O-78 0 86 12-299-2 O-78 0 87 12-299-3 O-78 0 88 12-324 O-78 0 8912-338-1 O-78 0 90 12-338-4 O-78 0

As shown in table 2, the bacteriophage ΦCJ24 exhibits infection abilityto avian pathogenic Escherichia coli (including O-1, O-78 serotypes),which are major causative bacteria of avian colibacillosis in generalpoultry farms.

It is known that O-78 serotype is generally the most dominant strainamong avian pathogenic Escherichia coli isolated from poultry farms.

The invention claimed is:
 1. A method of preparing an additivecomposition, the method comprising: providing bacteriophage ϕCJ24deposited as accession number KCCM11462P; and mixing the bacteriophagewith at least one additional material to provide the additivecomposition.
 2. The method of claim 1, wherein the bacteriophage is inan amount of 0.05 wt % to 10 wt % based on the weight of the additivecomposition.
 3. A method of preparing a feed composition, the methodcomprising: preparing the additive composition according to the methodof claim 1; and mixing the additive composition with an avian feed toprovide the feed composition.
 4. A method of feeding, the methodcomprising: preparing the feed composition according to the method ofclaim 3; and providing the feed composition to birds.
 5. A method ofpreparing drinking water composition, the method comprising: preparingthe additive composition according to the method of claim 1; and mixingthe additive composition with drinking water to provide the drinkingwater composition.
 6. A method of providing drinking water to birds, themethod comprising: preparing the drinking water composition according tothe method of claim 5; and providing the drinking water composition tobirds.